HER2 drives Mucin-like 1 to control proliferation in breast cancer cells.

Mucin-like 1 (MUCL1) was first identified as a breast-specific gene over a decade ago. Based on its highly restricted mRNA expression in breast tissue and continued expression during breast tumorigenesis and progression, MUCL1 is an attractive tumor-associated antigen and a potential therapeutic target. However, very little is known about the cellular location, biological functions and regulation of the MUCL1 protein, which will have a major impact on its druggability. Here we describe our efforts to fully characterize the cellular localization of MUCL1, investigate its regulation by key breast cancer oncogenes such as human epidermal growth factor receptor 2 (HER2) and discover its functional roles in breast cancer. Although some mucins are membrane bound, our data indicate that MUCL1 is secreted by some breast cancer cells, whereas others only express high levels of intracellular MUCL1. MUCL1 expression is highest in HER2-amplified breast tumors and inhibiting HER2 activity in tumor cells resulted in a decreased MUCL1 expression. In-depth investigation demonstrated that phosphoinositide3-kinase/Akt pathway, but not Ras/MEK pathway, controls MUCL1 expression downstream of HER2. Phenotypic assays revealed a strong dependence of HER2-positive cells on MUCL1 for cell proliferation. We further identified the mechanism by which MUCL1 regulates cell growth. Knockdown of MUCL1 induced a G1/S phase arrest concomitant with decreased cyclin D and increased p21 and p27 levels. Finally, we investigated the impact of MUCL1 loss on kinase signaling pathways in breast cancer cells through phospho-kinase array profiling. MUCL1 silencing abrogated phospho-focal adhesion kinase (FAK), Jun NH2-terminal kinase (JNK) and c-Jun signals, but not extracellular signal-regulated kinase or Akt pathway activities, thereby pointing to FAK/JNK pathway as the downstream effector of MUCL1 signaling. We are the first to identify an important role for MUCL1 in the proliferation of breast cancer cells, probably mediated via the FAK/JNK signaling pathway. Taken together, these data suggest a potential utility for therapeutic targeting of this protein in breast cancer.

A novel oncogenic role for the miRNA-506-514 cluster in initiating melanocyte transformation and promoting melanoma growth.

Malignant melanoma is the most aggressive form of skin cancer and its incidence has doubled in the last two decades. It represents only 4% of skin cancer cases per year, but causes as many as 74% of skin cancer deaths. Early detection of malignant melanoma is associated with survival rates of up to 90%, but later detection (stage III to stage IV) is associated with survival rates of only 10%. Dysregulation of microRNA (miRNA) expression has been linked to tumor development and progression by functioning either as a tumor suppressor, an oncogene or a metastasis regulator in multiple cancer types. To understand the role of miRNA in the pathogenesis of malignant melanoma and identify biomarkers of metastasis, miRNA expression profiles in skin punches from 33 metastatic melanoma patients and 14 normal healthy donors were compared. We identified a cluster of 14 miRNAs on the X chromosome, termed the miR-506-514 cluster, which was consistently overexpressed in nearly all melanomas tested (30-60 fold, P<0.001), regardless of mutations in N-ras or B-raf. Inhibition of the expression of this cluster as a whole, or one of its sub-clusters (Sub-cluster A) consisting of six mature miRNAs, led to significant inhibition of cell growth, induction of apoptosis, decreased invasiveness and decreased colony formation in soft agar across multiple melanoma cell lines. Sub-cluster A of the miR-506-514 cluster was critical for maintaining the cancer phenotype, but the overexpression of the full cluster was necessary for melanocyte transformation. Our results provide new insights into the functional role of this miRNA cluster in melanoma, and suggest new approaches to treat or diagnose this disease.

Axin-dependent phosphorylation of the adenomatous polyposis coli protein mediated by casein kinase 1epsilon.

Axin and the adenomatous polyposis coli protein (APC) interact to down-regulate the proto-oncogene beta-catenin. We show that transposition of an axin-binding site can confer beta-catenin regulatory activity to a fragment of APC normally lacking this activity. The fragment containing the axin-binding site also underwent hyperphosphorylation when coexpressed with axin. The phosphorylation did not require glycogen synthase kinase 3beta but instead required casein kinase 1epsilon, which bound directly to axin. Mutation of conserved serine residues in the beta-catenin regulatory motifs of APC interfered with both axin-dependent phosphorylation and phosphorylation by CKIepsilon and impaired the ability of APC to regulate beta-catenin. These results suggest that the axin-dependent phosphorylation of APC is mediated in part by CKIepsilon and is involved in the regulation of APC function.

Overexpression of the retinoic acid-responsive gene Stra6 in human cancers and its synergistic induction by Wnt-1 and retinoic acid.

Genetic defects in the Wnt-1 signaling pathway contribute to human tumor progression and are especially prevalent in colorectal cancer. We screened mouse C57MG cells to isolate mRNAs induced by Wnt-1 and identified Stra6, an mRNA known to be up-regulated by retinoic acid. Up-regulation of Stra6 mRNA was also observed in hyperplastic mammary tissue and mammary gland tumors from transgenic mice expressing Wnt-1 and in human tumors that frequently harbor defects in Wnt-1 signaling. Stimulation of C57MG cells with retinoic acid plus Wnt-1 resulted in expression of Stra6 transcript to levels greatly exceeding that observed with either stimulus alone. This synergy could be explained in part by the up-regulation of retinoic acid receptor-gamma that was observed in response to Wnt-1 signaling. Accordingly, treatment of human colorectal cancer cell lines with retinoic acid resulted in the up-regulation of Stra6 mRNA and accumulation of Stra6 protein at the cell membrane. The data support a model in which Wnt-1 signaling synergizes with retinoids to activate retinoic acid receptor-gamma-responsive genes in human cancers.

Src family kinases and HER2 interactions in human breast cancer cell growth and survival.

Evidence from murine fibroblast models and human breast cancer cells indicates that c-Src and human EGF receptor (HER1) synergize to enhance neoplastic growth of mammary epithelial cells. To investigate whether interactions between c-Src and other HER family members may also play a role in breast tumor progression, we characterized 13 human breast carcinoma cell lines and 13 tumor samples for expression of HER family members and c-Src and examined a subset of the cell lines for Src-dependent, heregulin (HRG)-augmented, anchorage-dependent and independent growth. By immunoblotting, we found that all cell lines overexpressed one or more HER family member, and 60% overexpressed c-Src. Seventy-five per cent of the tumor tissues overexpressed HER2, while 64% overexpressed c-Src. Colony formation in soft agar was enhanced by HRG in three of five cell lines tested, a response that correlated with the presence of a c-Src/HER2 heterocomplex. This result suggests that HRG may act through both HER2 and c-Src to facilitate anchorage-independent growth. In contrast, HRG had little effect on anchorage-dependent growth in any of the cell lines tested. PP1, a Src family kinase inhibitor, reduced or ablated HRG-dependent and independent soft agar growth or anchorage dependent growth, and triggered apoptosis in all cell lines tested. The apoptotic effect of PP1 could be partially or completely reversed by HRG, depending on the cell line. These results suggest that while Src family kinases may cooperate with HRG to promote the survival and growth of human breast tumor cells, they also function independently of HER2/HRG in these processes.

Practice guidelines for lipid-based amphotericin B in stem cell transplant recipients.

OBJECTIVE: To provide clinicians who practice in the stem cell transplantation (SCT) setting with practical guidelines for the use of lipid-based amphotericin B (AmB) formulations in SCT patients who have documented or probable invasive fungal infections, are experiencing neutropenic fever, or require secondary prophylaxis for fungal infections. DATA SOURCES: Recommendations are based on the results of a two-day consensus meeting that convened clinicians versed in the management of infectious complications in patients undergoing SCT. This meeting, which was held October 21-23, 1998, in Orlando, Florida, was sponsored by an educational grant from The Liposome Company. In addition, primary articles were identified by MEDLINE search (1980-December 1999) and through secondary sources. STUDY SELECTION AND DATA EXTRACTION: All of the articles identified from the data sources were evaluated, and all information deemed relevant was included in this review. DATA SYNTHESIS: Immunocompromised patients, particularly patients undergoing high-dose chemotherapy with SCT, experience a high degree of morbidity and mortality from invasive fungal infections. Historically, treatment for such infections with conventional AmB had been limited primarily by its associated nephrotoxicity. Lipid-based formulations of AmB have helped to advance the management of invasive fungal infections in the SCT population by offering a treatment alternative that allows for administration of adequate amounts of active drug to produce clinical and mycologic responses, compared with conventional AmB, in a delivery system that is less nephrotoxic. Unfortunately, these agents are relatively expensive. Therefore, patients who are candidates for lipid-based products must be selected carefully. CONCLUSIONS: Practical guidelines are provided for the use of lipid-based AmB formulations in SCT patients who have documented or probable invasive fungal infections, are experiencing neutropenic fever, or require secondary prophylaxis for fungal infections.

Emotional distress regulation takes precedence over impulse control: if you feel bad, do it!

Why do people's impulse controls break down during emotional distress? Some theories propose that distress impairs one's motivation or one's ability to exert self-control, and some postulate self-destructive intentions arising from the moods. Contrary to those theories, Three experiments found that believing that one's bad mood was frozen (unchangeable) eliminated the tendency to eat fattening snacks (Experiment 1), seek immediate gratification (Experiment 2), and engage in frivolous procrastination (Experiment 3). The implication is that when people are upset, they indulge immediate impulses to make themselves feel better, which amounts to giving short-term affect regulation priority over other self-regulatory goals.

If you can't join them, beat them: effects of social exclusion on aggressive behavior.

Social exclusion was manipulated by telling people that they would end up alone later in life or that other participants had rejected them. These manipulations caused participants to behave more aggressively. Excluded people issued a more negative job evaluation against someone who insulted them (Experiments 1 and 2). Excluded people also blasted a target with higher levels of aversive noise both when the target had insulted them (Experiment 4) and when the target was a neutral person and no interaction had occurred (Experiment 5). However, excluded people were not more aggressive toward someone who issued praise (Experiment 3). These responseswere specific to social exclusion (as opposed to other misfortunes) and were not mediated by emotion

Longitudinal improvement of self-regulation through practice: building self-control strength through repeated exercise.

This study examined the results of repeated exercises of self-control in relation to self-regulatory strength over time. A sample of 69 U.S. college students spent 2 weeks doing 1 of 3 self-control exercises: monitoring and improving posture, regulating mood, or monitoring and recording eating. Compared with a no-exercise control group, the participants who performed the self-control exercises showed significant improvement in self-regulatory capacity as measured by quitting faster on a hand-grip exercise task following a thought-suppression exercise.

c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function.

Accumulating evidence indicates that interactions between the epidermal growth factor receptor (EGFR) and the nonreceptor tyrosine kinase c-Src may contribute to an aggressive phenotype in multiple human tumors. Previous work from our laboratory demonstrated that murine fibroblasts which overexpress both these tyrosine kinases display synergistic increases in DNA synthesis, soft agar growth, and tumor formation in nude mice, and increased phosphorylation of the receptor substrates Shc and phospholipase gamma as compared with single overexpressors. These parameters correlated with the ability of c-Src and EGFR to form an EGF-dependent heterocomplex in vivo. Here we provide evidence that association between c-Src and EGFR can occur directly, as shown by receptor overlay experiments, and that it results in the appearance of two novel tyrosine phosphorylations on the receptor that are seen both in vitro and in vivo following EGF stimulation. Edman degradation analyses and co-migration of synthetic peptides with EGFR-derived tryptic phosphopeptides identify these sites as Tyr845 and Tyr1101. Tyr1101 lies within the carboxyl-terminal region of the EGFR among sites of receptor autophosphorylation, while Tyr845 resides in the catalytic domain, in a position analogous to Tyr416 of c-Src. Phosphorylation of Tyr416 and homologous residues in other tyrosine kinase receptors has been shown to be required for or to increase catalytic activity, suggesting that c-Src can influence EGFR activity by mediating phosphorylation of Tyr845. Indeed, EGF-induced phosphorylation of Tyr845 was increased in MDA468 human breast cancer cells engineered to overexpress c-Src as compared with parental MDA 468 cells. Furthermore, transient expression of a Y845F variant EGFR in murine fibroblasts resulted in an ablation of EGF-induced DNA synthesis to nonstimulated levels. Together, these data support the hypothesis that c-Src-mediated phosphorylation of EGFR Tyr845 is involved in regulation of receptor function, as well as in tumor progression.

Mechanism of biological synergy between cellular Src and epidermal growth factor receptor.

Overexpression of both cellular Src (c-Src) and the epidermal growth factor receptor (EGFR) occurs in many of the same human tumors, suggesting that they may functionally interact and contribute to the progression of cancer. Indeed, in murine fibroblasts, overexpression of c-Src has been shown to potentiate the mitogenic and tumorigenic capacity of the overexpressed EGFR. Potentiation correlated with the ability of c-Src to physically associate with the activated EGFR and the appearance of two unique in vivo phosphorylations on the receptor (Tyr-845 and Tyr-1101). Using stable cell lines of C3H10T1/2 murine fibroblasts that contain kinase-deficient (K-) c-Src and overexpressed wild-type EGFR, we show that the kinase activity of c-Src is required for both the biological synergy with the receptor and the phosphorylations on the receptor, but not for the association of c-Src with the receptor. In transient transfection assays, not only epidermal growth factor but also serum- and lysophosphatidic acid-induced DNA synthesis was ablated in a dominant-negative fashion by a Y845F mutant of the EGFR, indicating that c-Src-induced phosphorylation of Y845 is critical for the mitogenic response to both the EGFR and a G protein-coupled receptor (lysophosphatidic acid receptor). Unexpectedly, the Y845F mutant EGFR was found to retain its full kinase activity and its ability to activate the adapter protein SHC and extracellular signal-regulated kinase ERK2 in response to EGF, demonstrating that the mitogenic pathway involving phosphorylation of Y845 is independent of ERK2-activation. The application of these findings to the development of novel therapeutics for human cancers that overexpress c-Src and EGFR is discussed.

Prolongation of porcine islet xenograft survival in mice after therapy with immunosuppressive peptides.

BACKGROUND: Recently, peptides derived from the heavy chain of HLA-B2702 have been shown to modulate immune responses. In this study, we examined the use of these peptides for immunosuppression in a pig to mouse islet xenograft model. METHODS: Purified porcine islets were transplanted in autoimmune (non-obese diabetic) and non-autoimmune (streptozotocin-injected CBA or C57/Bl6) diabetic mice. Various dosing regimens of HLA-derived peptides with and without antilymphocyte therapy were administered to recipient mice. Graft rejection was determined by daily serum glucose determinations, and, at selected time points, grafts were removed to demonstrate function and provide immunohistochemical examination. RESULTS: HLA-derived peptides were demonstrated to prolong graft survival in both pretransplant and posttransplant treatment regimens. This effect was increased with concomitant antilymphocyte therapy. CONCLUSIONS: Further elucidation of the mechanism of action of these immunomodulatory peptides may help in the development of novel immunosuppressive protocols.

Ego depletion: is the active self a limited resource?

Choice, active response, self-regulation, and other volition may all draw on a common inner resource. In Experiment 1, people who forced themselves to eat radishes instead of tempting chocolates subsequently quit faster on unsolvable puzzles than people who had not had to exert self-control over eating. In Experiment 2, making a meaningful personal choice to perform attitude-relevant behavior caused a similar decrement in persistence. In Experiment 3, suppressing emotion led to a subsequent drop in performance of solvable anagrams. In Experiment 4, an initial task requiring high self-regulation made people more passive (i.e., more prone to favor the passive-response option). These results suggest that the self's capacity for active volition is limited and that a range of seemingly different, unrelated acts share a common resource.

Self-control as limited resource: regulatory depletion patterns.

If self-regulation conforms to an energy or strength model, then self-control should be impaired by prior exertion. In Study 1, trying to regulate one's emotional response to an upsetting movie was followed by a decrease in physical stamina. In Study 2, suppressing forbidden thoughts led to a subsequent tendency to give up quickly on unsolvable anagrams. In Study 3, suppressing thoughts impaired subsequent efforts to control the expression of amusement and enjoyment. In Study 4, autobiographical accounts of successful versus failed emotional control linked prior regulatory demands and fatigue to self-regulatory failure. A strength model of self-regulation fits the data better than activation, priming, skill, or constant capacity models of self-regulation.

Overexpression of cellular Src in fibroblasts enhances endocytic internalization of epidermal growth factor receptor.

Previous studies have demonstrated a requirement for the nonreceptor tyrosine kinase, cellular Src (c-Src), in epidermal growth factor (EGF)-induced mitogenesis and a synergistic interaction between c-Src and EGF receptor (EGFR) in tumorigenesis. Although endocytic internalization of EGFR may be thought to attenuate EGF-stimulated signaling, recent evidence suggests that signaling through Ras can be amplified by repeated encounters of endosome-localized, receptor. Shc.Grb2.Sos complexes with the plasma membrane, where Ras resides almost exclusively. Based on these reports, we examined EGFR trafficking behavior in a set of single and double c-Src/EGFR C3H10T1/2 overexpressors to determine if c-Src affects basal receptor half-life, ligand-induced internalization, and/or recycling. Our results show that overexpression of c-Src causes no change in EGFR half-life but does produce an increase in the internalization rate constant of EGF.EGFR complexes when the endocytic apparatus is not stoichiometrically saturated; this effect of c-Src on EGFR endocytosis is negligible at high receptor occupancy in cells overexpressing the receptor. In neither case are EGFR recycling rate constants affected by c-Src. These data indicate a functional role for c-Src in receptor internalization, which in turn could alter some aspects of EGFR signaling related to mitogenesis and tumorigenesis.

Survival of rat small bowel allografts treated with allotrap 07R.

Previous reports from other investigators demonstrate prolongation of allogeneic heart graft survival and decrease in CTL responses in rats treated with a small synthetic peptide corresponding to residues 75-84 of the human HLA-B7-01 molecule (Allotrap 07R). We wished to determine the efficacy of these peptides in the highly immunogenic ACI > LEW and LEW > ACI small bowel transplant models. Animals were divided into treatment groups: I, none; II, Allotrap (20 mg/kg/day on Days 0-4); III, cyclosporine (CsA; 10 mg/kg/day on Days 0-4); IV, Allotrap + CsA (as in groups II and III); V, Allotrap (40 mg/kg/day every other day on Days -19 to 4); VI, Allotrap + CsA (as in groups III and V); VII, Allotrap + CsA (as in groups III and V, with Allotrap administered intragraft Days 0-4). The animals were sacrificed at the time of graft rejection (defined by dusky, necrotic stoma and increased stomal output). Peripheral blood, spleen, native bowel, and allograft intraepithelial and lamina propria lymphocytes were harvested and mixed lymphocyte culture (MLC) reactivity against self, donor, and third-party splenocytes was assessed. Statistical analysis was performed by ANOVA with Dunnett's t for multiple comparisons against a control as a post hoc test. We found a very slight, but significant prolongation of graft survival in with treatment protocol V for both strain combinations. In addition, MLC response of splenocytes to donor antigen was decreased with combined CsA and Allotrap, but not with Allotrap alone. We conclude that Allotrap decreases response to alloantigens, and slightly, but significantly prolongs graft survival in the hihgly immunogenic small bowel transplant model.

Role of nitric oxide and tumor necrosis factor on lung injury caused by ischemia/reperfusion of the lower extremities.

PURPOSE: Acute aortic occlusion with subsequent ischemia/reperfusion (I/R) of the lower extremities is known to predispose to lung injury. The pathophysiologic mechanisms of this injury are not clear. In the present study, we studied the role of tumor necrosis factor (TNF) and nitric oxide (NO) in lung injury caused by lower extremity I/R. METHODS: A rat model in which the infrarenal aorta was cross-clamped for 3 hours followed by 1 hour of reperfusion was used. The rats were randomized into five groups: group 1, aorta exposed but not clamped; group 2, aorta clamped for 3 hours, followed by 1 hour of reperfusion; group 3, 1 mg/kg dexamethasone administered before the aorta was clamped; group 4, 25 mg aminoguanidine, a specific inducible NO synthase (iNOS) inhibitor, administered before the aorta was clamped; and group 5, 2 mg/kg TNFbp, a PEG-ylated dimeric form of the high-affinity p55 TNF receptor I (RI), administered before the aorta was clamped. NO concentration in the exhaled gas (ENO) was measured, as an index of NO production by the lung, in 30 minute intervals during I/R. Serial arterial blood samples for TNF assay were obtained during the course of the experiment. At the end of the experiment, the lungs were removed and histologically examined for evidence of injury. RESULTS: ENO in group 2 increased from 0.7 +/- 0.3 ppb at baseline to 54.3 +/- 7.5 ppb at the end of ischemia and remained stable during reperfusion (54.6 +/- 8.5 ppb at the end of reperfusion). ENO production was blocked by aminoguanidine, by dexamethasone, and by TNFbp given before aortic occlusion. Serum TNF in groups 2, 3 and 4 increased rapidly during early ischemia, reaching its peak value 60 minutes after occlusion of the aorta, then gradually declined to baseline levels at the end of ischemia, and remained low during reperfusion. TNFbp decreased serum TNF concentration significantly when it was given before aortic occlusion. Histologic examination of the lungs at the end of the experiment revealed that aminoguanidine, dexamethasone, and TNFbp had a protective effect on the lungs. CONCLUSIONS: Serum TNF increases rapidly during lower extremity ischemia and causes increased production of NO from the lung by upregulating iNOS. Increased NO is associated with more severe lung injury, and iNOS blockade has beneficial effects on the lung. TNF blockade before ischemia decreases NO production by the lung and attenuates lung injury. ENO can be used as an early marker of lung injury caused by lower extremity I/R.